Benzyl all-trans-retinoate used for the treatment of acne

ABSTRACT

Esters and amides of all-trans-retinoic acid are disclosed which are useful for the treatment of acne.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of our copending application Ser. No.628,185, filed Nov. 3, 1975, now U.S. Pat. No. 4,055,659, issued Oct.25, 1977.

BACKGROUND OF THE INVENTION

Retinoic acid is known to be useful for the treatment of acne. See, forexample, U.S. Pat. No. 3,729,568, which discloses the use of retinoicacid compositions for the treatment of acne. Side effects associatedwith the use of retinoic acid, such as, for example, excessive erythema,have stimulated efforts to discover other acne treatment materials whichwould not have these side effects.

In order to discover such materials, the well-known ability of retinoicacid to promote increased epidermal DNA synthesis upon topicalapplication was utilized. This ability has been associated with theeffectiveness of retinoic acid in the treatment of acne. See, forexample, Christophers and Braun-Falco: Stimulation of EpidermalDNA-Synthesis with Vitamin A-Acid, Arch. Klin. exp. Derm. 232: 427-433(1968) and Wolfe, et al.: Changes in Epidermal Differentiation AfterVitamin A Acid, Arch. Klin. exp. Derm. 237: 744-795 (1970). Forty-eightnovel esters and amides of all-trans-retinoic acid have been preparedand tested for their efficacy in promoting increased epidermal DNAsynthesis.

SUMMARY OF THE INVENTION

This invention relates to esters and amides of all-trans-retinoic acidand more particularly to those esters and amides having the formula:##STR1## wherein X is a member selected from the group consisting of:--OCH₂ CONH₂ ; mixed --OCH₂ CH(OH)CH₃ and --OCH(CH₃)CH₂ OH; --OCH₂ CH₂OH; ##STR2##

It has surprisingly been discovered that only these seven novelcompounds, of the 48 novel compounds prepared and tested, are active inthe following test measuring DNA synthesis and therefore are useful forthe treatment of acne. Further, these seven novel compounds generally donot produce the excessive erythema characteristic of retinoic acid.While it is believed that these compounds are useful for the treatmentof acne, it is also suggested that they are useful for treatment oramelioration of the same additional classes of skin disorders as isretinoic acid itself. These disorders include ichthyoses (e.g.,ichthyosis hystrix, epidermolytic hyperkeratosis, and lamelarichthyosis), follicular disorders (e.g., pseudolfolliculites, senilecomedones, nevus comidonicus, and trichostatis spinulosa), benignepithelial tumors (e.g., flat warts, trichoepithelioma, and molluscumcontagiosum), perforated dermatoses (e.g., elastosis perforansserpiginosa and Kyrle's disease), and disorders of keratinization (e.g.Darier's disease, keratoderma, hyperkeratosis plantaris, pityriasisrubra pilaris, lichen planus, acanthosis nigricans, and psoriosis).

Ten male guinea pigs (Hartley strain) weighing ca. 400 g each were usedfor each compound to be tested. The animals were housed singly in wirecages, handled daily during experimentation, provided chow and water adlibitum, and maintained on 12/12 hour light/dark cycles. Prior to theexperimental procedure, the animals were maintained as just describedfor 3 days.

On the first day of the test, one ear (dorsal skin) of each animal wasrandomly selected and treated with 0.025 ml of the experimentalsolution, and the other ear was treated with an equal volume of theplacebo vehicle (control). Ten animals for each compound were sotreated. These topical applications were made at 9:00 am on the first 4days of the experiment. All the animals received chronic administrationof tritiated thymidine (³ H-TdR) for these first four days. The ³ H-TdRwas given intraperitoneally at about 9:30 am, 1:30 pm and 5:00 pm ofeach day (10 micro Curie in 0.1 ml H₂ O/injection; specific activity=2.0Curie/millimole). On the fifth day (9:00 am), the animals were killed,and 6 mm diameter punches of ear skin from the central portion of thetreated sites were harvested.

Each of these tissue samples was solubilized at 37°-50° C. for 13 daysin 1 ml of an alkali solubilizer sold by Amersham-Searle Co. under theregistered trademark "NCS" Solubilizer. The dissolved tissues kept innylon scintillation vials were acidified with 0.025-0.050 ml of aceticacid, and diphenyloxazole in toluene was added as a fluor to detect thebeta particles emitted by absorbed tritiated thymidine. Theradioactivity of the samples was determined by multiple counting on aBeckman LS counter. All count per minute values were quench corrected byexternal standardization to yield disintegrations per minute (dpm)/6 mmpunch of skin. Quench correction is especially important as some of thesamples show faint yellow coloration due to presence of the retinylderivative, and therefore present considerable color quench.

The test compounds were applied as dilute solutions (0.05%-0.2%) in50:50 parts by weight propylene glycolethanol or 35:35:30 parts byweight propylene glycol-ethanolchloroform. The compounds of theinvention provoke thymidine uptake significantly greater than thecontrol at greater than 90% confidence level and generally at greaterthan 99% confidence level. The test results for the compounds of theinvention are shown in Table I, to which results for unsubstitutedall-trans-retinoic acid are added for comparison.

                                      TABLE I                                     __________________________________________________________________________    COMPOUND                 DPM (standard error)                                                                     Confidence Level                          __________________________________________________________________________    0.05%                                                                             all-trans-retinoyloxyacetamide                                                                     2360                                                                              (408)     90%                                        Control              1779                                                                              (211)                                            0.05%                                                                             mixed 2-hydroxy-1-propyl and 1-hydroxy-2-                                     propyl-all-trans-retinoates                                                                        1672                                                                              (152)     99%                                        Control              1259                                                                              (91)                                             0.2%                                                                              2-hydroxyethyl-all-trans-retinoate                                                                 12945                                                                             (1669)    99.9%                                      Control              3416                                                                              (947)                                            0.2%                                                                              N-(3,4-methylenedioxyphenylmethyl)-all-                                       trans-retinamide     11,186                                                                            (2266)    99%                                        Control              2862                                                 0.2%                                                                              4-nitrobenzyl all-trans-retinoate                                                                  13910                                                                             (1252)    99.9%                                      Control              1906                                                                              (158)                                            0.2%                                                                              benzyl all-trans-retinoate                                                                         14168                                                                             (2346)    99%                                        Control              2006                                                                              (418)                                            0.05%                                                                             4-(all-trans-retinoyloxyacetyl)-catechol                                                           2116                                                                              (275)     99%                                        Control              1230                                                                              (83)                                             0.05%                                                                             all-trans-retinoic acid                                                                            4429                                                                              (912)     99%                                        Control              1504                                                                              (147)                                            __________________________________________________________________________

Surprisingly, only these seven compounds of the 48 novel compoundstested gave positive results on this test and hence are useful for thetreatment of acne. The compounds of the invention may be topicallyapplied to the acne site in any suitable pharmaceutically-acceptablevehicle, as for example a liquid carrier such as propyleneglycol-ethanol, propylene glycol-ethanol-chloroform, and the like. Apreferred liquid composition is a solution of a small amount of at leastone of the compounds of the invention in a combination of (A) from about25% to about 75% by weight of 95% ethanol and (B) from about 75% toabout 25% by weight of a liquid glycol. A typical solvent carrier ofthis type comprises 50% by weight 95% ethyl alcohol and 50% by weightpropylene glycol. The preferred concentration of the active compound inthese compositions is at least about 0.01% by weight, more preferablyfrom about 0.01% to about 0.5% by weight, and most preferably from about0.5% to about 0.2% by weight, but any therapeutically effectiveconcentration may be used. This method of use is the same as taught inthe above-mentioned U.S. Pat. No. 3,729,568, the contents of which areincorporated herein by reference. These compositions and the method oftreating acne by topical application to the acne site of at least one ofthe compounds of the invention are also considered part of the presentinvention.

The 41 novel esters and amides of all-trans-retinoic acid which wereinactive in the above test are listed below:

2-Cyclohexylethyl all-trans-Retinoate

10-Carbomethoxydecyl all-trans-Retinoate

4-Hydroxybutyl all-trans-Retinoate

Cholesteryl all-trans-Retinoyloxyacetate

Cholesteryl all-trans-Retinoate

Mixed m-and p-vinylbenzyl all-trans-Retinoates

4-Bromobenzyl all-trans-Retinoate

all-trans-Retinoyloxyacetylbenzene

4-(all-trans-Retinoyloxyacetyl)-bromobenzene

4-(all-trans-Retinoyloxyacetyl)-methoxybenzene

4-(all-trans-Retinoyloxyacetyl)-nitrobenzene

4-(all-trans-Retinoyloxyacetyl)-phenol

4-(all-trans-Retinoyloxyacetyl)-toluene

4-(all-trans-Retinoyloxyacetyl)-benzonitrile

4-(all-trans-Retinoyloxyacetyl)-ethoxybenzene

4-(all-trans-Retinoyloxyacetyl)-acetoxybenzene

4-(all-trans-Retinoyloxyacetyl)-naphthalene

4-(all-trans-Retinoyloxyacetyl)-biphenyl

all-trans-Retinoyloxyacetyl-2,5-dimethoxybenzene

all-trans-Retinoyloxyacetyl-2,4-dichlorobenzene

all-trans-Retinoyloxyacetyl-2,4-dimethylbenzene

all-trans-Retinoyloxyacetyl-3,4-diacetoxybenzene

all-trans-Retinoyloxyacetyl-3,4,5-trimethoxybenzene

all-trans-Retinoyloxyacetyl-2,4,6-trimethylbenzene

N-n-Propyl all-trans-retinamide

N-tert-Butyl all-trans-retinamide

N-(1,1,3,3-tetramethyl)-butyl all-trans-retinamide

N-(all-trans-Retinoyl)-morpholine

4-(all-trans-Retinoyl)-aminophenol

Methyl 4-(all-trans-Retinoylamino)-salicylate

N,N'-Dicyclohexyl-N-(all-trans-retinoyl)-urea

Acetone (all-trans-Retinoyl)-hydrazone

trans-β-Ionone (all-trans-Retinoyl)-hydrazone

N-(all-trans-Retinoyl)-imidazole

N-(all-trans-Retinoyloxy)-succinimide

N-(all-trans-Retinoyloxy)-phthalimide

1-Nicotinoyl-2-(all-trans-retinoyl)-hydrazine

1-(all-trans-Retinoyl)-benzotriazole

1-(all-trans-Retinoyl)-1,2,4-triazole

N-[β-(3,4-Dimethoxyphenyl)ethyl]-all-trans-retinamide

2-(all-trans-Retinoylamino)-benzothiazole

These forty-one inactive compounds are the subject of our United StatesPatent Application entitled "Retinoic Acid Derivatives," Ser. No.628,177 filed on Nov. 3, 1975.

The preparation of the compounds of the present invention is illustratedby the following examples.

EXAMPLE I all-trans RETINOYLOXYACETAMIDE

In a 1000 ml 3-necked round-bottom flask equipped with reflux condenser,stirrer, and nitrogen inlet were placed ethyl acetate (500 ml),all-trans-retinoic acid (10 g; 0.30 mole), anhydrous potassium carbonate(4.6 g, 0.30 mole), 2-chloroacetamide (30 g, 0.32 mole) and sodiumiodide (5.0 g, 0.30 mole). This mixture was stirred at refluxtemperature for eighteen hours under nitrogen atmosphere.

At the end of the eighteen-hour period, the mixture was cooled to roomtemperature and extracted with three 500-ml portions of water, three500-ml portions of 10% sodium carbonate in water, and one 500-ml portionof water. After each extraction the upper layer was retained and thelower layer discarded.

The final upper layer was dried over anhydrous magnesium sulfate,filtered, and the filtrate evaporated under reduced pressure, leaving anorange oil. The oil was dried at ambient temperature and 0.5 mmpressure, causing it to turn to an orange-yellow solid, melting at62°-64° C.

The crude product was recrystalized from hot toluene yielding pureall-trans-retinoyloxyacetamide, melting at 133°-134° C. The protonmagnetic resonance spectrum of the compound was consistent with thestructure written.

Anal. Calcd. for C₂₂ H₃₁ NO₃ : C, 74.4 H, 8.2 N, 3.9. Found: C, 74.4 H,8.5 N. 3.9.

EXAMPLE II MIXED 2-HYDROXY-1-PROPYL and 1-HYDROXY-2-PROPYLall-trans-RETINOATES

A U.S.P. grade propylene glycol (1,2-propanediol) was used in thispreparation. A solution of 6 ml of propylene glycol and 3 ml of pyridinein 10 ml of benzene reacted readily when added during about threeminutes to a solution of retinoyl chloride (which had been prepared from3.0 g of all-trans-retinoic acid and phosphorous trichloride in 25 ml ofbenzene according to directions of Huisman, et al., Rec. trav. chim.,75, 977-1006 (1956)). The reaction was moderated at or below roomtemperature with a Dry Ice-isopropanol bath. The reaction mixture wasstirred magnetically under a nitrogen atmosphere at room temperature foran hour, during which time the reaction flask was kept covered withaluminum foil.

The golden-yellow reaction solution was diluted with 20 ml of ethylether and washed with two 20-ml portions of 2% hydrochloric acid, with20 ml of 10% aqueous sodium bicarbonate solution, and finally with 20 mlof 10% saturated sodium chloride solution. The washed benzene-ethersolution was dried over anhydrous magnesium sulfate.

The solution was then placed on 75 ml of activated alumina in achromatography column. The column was eluted with 2 liters of ethylether, the composition of the eluates being monitored by thin layerchromatography. Impurities were eluted in the early fractions. Laterfractions containing product were combined, and the solvent wasevaporated in a rotary evaporator under vacuum. The residue was a yellowoil which was crystallized from n-hexane to yield a solid. Tworecrystallizations gave yellow plates, melting point 91°-95° C. Theultraviolet absorption spectrum measured in isopropanol showed a maximumat 353 nm. The proton magnetic resonance spectrum indicated the mixedisomers, ca. 70% 2-hydroxypropyl-all-trans-retinoate and ca. 30%1-hydroxypropyl-all-trans-retinoate.

Anal. Calcd. for C₂₃ H₃₃ O₃ : C, 77.2 H, 9.30. Found: C, 77.2 H, 9.47.

EXAMPLE III 2-HYDROXYETHYL all-trans-RETINOATE

A solution of retinoyl chloride was prepared from 3.0 g ofall-trans-retinoic acid and 0.92 g of phosphorus trichloride in 25 ml ofbenzene. This solution was added gradually to a solution of 6 ml ofethylene glycol and 8 ml of pyridine during a period of 40 minutes. Themoderate reaction exotherm was controlled by cooling the reaction flaskin an ice bath. The reaction was then stirred under a nitrogenatmosphere for 30 minutes longer.

The orange reaction mixture was placed directly on 80 ml of activatedalumina in a chromatographic column, and the column was eluted with 1.4liters of anhydrous ethyl ether. The ether was evaporated and theresidual yellow oil was applied to the top of a fresh column of 80 ml ofalumina. This column was eluted successively with 1 liter of ether and0.5 liter of ethyl acetate. Composition of the eluate was monitored bythin layer chromatography.

Evaporation of the latter fractions yielded a crude yellow crystallineproduct. Recrystallization, first from 5 ml n-hexane, then from 10 ml95:5 n-hexane-benzene gave yellow plates, melting point 108°-110° C. Theproton magnetic resonance spectrum verified the structure of thecompound.

ANAL. Calcd. for C₂₂ H₃₂ O₃ : C, 76.7 H, 9.37. Found: C, 76.7 H, 9.28.

EXAMPLE IV N-(3,4-METHYLENEDIOXYPHENYLMETHYL)-all-trans-RETINAMIDE

A solution of retinoyl chloride was prepared by magnetically stirring3.00 g of all-trans-retinoic acid and 0.92 g of phosphorus trichloridefor two hours in 50 ml of dry benzene. During 18 minutes the retinoylchloride was added to a solution of 7.55 g of piperonylamine in 10 ml ofanhydrous ethyl ether while stirring under a nitrogen atmosphere andcooling in an ice bath. Stirring was then continued for three hours atroom temperature, and for an hour more at 50° C.

The reaction mixture was diluted with 250 ml of ethyl ether. The ethersolution was extracted with two 25-ml portions of 5% hydrochloric acid,then washed with four 25-ml portions of cold water. After drying of thewashed solution over sodium sulfate, the solvent was evaporated leaving3.65 g of a sticky yellow solid. The solid hardened to a crystallinematerial when triturated with 50 ml of petroleum ether (b.p. 30°-60°C.). The crystals were filtered, washed with 25 ml of petroleum ether,and vacuum dried at room temperature to yield crude product. Thisproduct was recrystallized twice from methanol (5 ml per gram) givingpure compound, melting point 106°-107° C. The proton magnetic resonancespectrum was consistent with the structure with no extraneousresonances.

Anal. Calcd. for C₂₈ H₃₅ NO₃ : C, 77.6 H, 8.14 N, 3.23. Found: C, 77.6H, 8.15 N, 3.31.

EXAMPLE V 4-NITROBENZYL all-trans-RETINOATE

Potassium retinoate was made by neutralizing 3.00 g ofall-trans-retinoic acid in 30 ml of tetrahydrofuran with 15.6 ml of0.640 N methanolic potassium hydroxide. The solvent was evaporated on arotary evaporator and the residue dried at a pressure of less than 0.5mm. for several hours.

Powdered potassium retinoate (3.03 g) in 20 ml ofhexamethylphosphoramide was stirred overnight at room temperature with2.16 g of 4-nitrobenzyl bromide. The reaction mixture was poured into 30ml of cold 5% hydrochloric acid, precipitating a bright-yellow gum. Thegum was washed by decantation with 60 ml of cold water, then wasdissolved in 300 ml of ethyl ether. The ether solution was washed withfour 40-ml portions of ice water and dried over sodium sulfate.Evaporation of the ether left a bright-yellow powder.

The material was recrystallized twice from 50:50 chloroform-methanol (6ml per gram), giving tiny, bright-yellow prisms, melting point 130°-131°C. The proton magnetic resonance spectrum was consistent with thestructure.

Anal. Calcd. for C₂₇ H₃₃ NO₄ : C, 74.4 H, 7.63 N, 3.22. Found: C, 74.3H, 7.73 N, 3.14.

EXAMPLE VI BENZYL all-trans-RETINOATE

Powdered potassium retinoate (3.03 g) in 20 ml ofhexamethylphosphoramide was stirred overnight at room temperature with1.26 g of benzyl chloride. The reaction mixture was poured into 30 ml ofcold 5% hydrochloric acid, precipitating an oil which was then taken upin 150 ml of ethyl ether. The solution was washed with four 25-mlportions of ice water and dried over sodium sulfate. Evaporation of theether left a residue of yellow semi-solid. This residue was trituratedwith 50 ml of petroleum ether (b.p. 30°-60° C.) and chilled in ice. Thesolution was filtered from a little retinoic acid, and the filtrate wasthen evaporated to about 5 ml volume.

A chromatography tube 16×180 mm was filled to a depth of 125 mm withchromatographic silica gel, 40-140 mesh. The petroleum ether solution (5ml) was put on the column and the chromatogram developed with 50:50benzene-petroleum ether by volume. Material coming off the column wasmonitored by thin layer chromatography on silica gel plates using 70:30benzene-n-hexane developer and iodine vapor visualization. Evaporationof the purest fractions gave a mobile, clear, yellow oil.

The whole preparation was repeated and the product combined with the1.50 g from the first preparation. The combined material waschromatographed again as before. The residue after evaporation of thesolvent was dried by rotating it on a rotary evaporator at 0.2 mm forfour hours. The compound was a clear yellow liquid which exhibited thecorrect proton magnetic resonance spectrum and elemental analysis.

Anal. Calcd. for C₂₇ H₃₄ O₂ : C, 83.0 H, 8.77. Found: C, 83.3 H, 8.89.

EXAMPLE VII 4-(all-trans-RETINOYLOXYACETYL)-CATECHOL

Powdered potassium retinoate (3.03 g) and 1.86 g ofα-chloro-3',4'-dihydroxyacetophenone in 22 ml of hexamethylphosphoramidewere stirred overnight at 40°-45° C. The reaction mixture was pouredinto 30 ml of cold 5% hydrochloric acid, precipitating a thick yellowgum. The acid was decanted, and the gum was triturated with 30 ml ofdistilled water. The gum was then dissolved in 150 ml of ethyl ether andwashed with four 25-ml portions of cold water. The ether solution wasevaporated, leaving a dry, yellow powder.

The powder was recrystallized twice from 3:1 methanol-water by volume(42 ml per gram). Product was a yellow-green powder, melting point177°-178° C. The proton magnetic resonance spectrum was consistent withthe structure.

Anal. Calcd. for C₂₈ H₃₄ O₅ : C, 74.6 H, 7.61. Found: C, 74.0 H, 7.48.

The following example illustrates the use of the compounds of theinvention. All parts are by weight.

EXAMPLE VIII

    ______________________________________                                                            Parts                                                     ______________________________________                                        A liquid vehicle consisting of:                                               95% ethyl alcohol     500                                                     propylene glycol      500                                                     ______________________________________                                    

is prepared by mixing the ingredients together. To the resulting vehicleis added 2 parts of 2-hydroxyethyl-all-trans-retinoate, and the whole isthoroughly mixed until the active ingredient is dissolved. The resultingcomposition is useful for the treatment of acne by periodic topicalapplication to the acne site. Application generally should be made atleast once daily for at least one month.

The above examples have been provided to illustrate the presentinvention, the scope of which is defined by the following claims.

What is claimed is:
 1. A pharmaceutical composition for the treatment ofacne by topical application which comprises an effective acne-treatmentamount of benzyl all-trans-retinoate admixed with a pharmaceuticallyacceptable topical vehicle which is a mixture selected from the groupconsisting of propylene glycol-ethanol and propyleneglycol-ethanol-chloroform.
 2. A method for treatment of acne in asubject requiring such treatment which comprises topical application tothe acne site of said subject of a composition comprising an effectiveacne-treatment amount of benzyl all-trans-retinoate admixed with apharmaceutically-acceptable topical vehicle.
 3. The method of claim 2wherein the benzyl all-trans-retinoate comprises from about 0.01% toabout 0.5% by weight of the composition.
 4. The method of claim 3wherein the benzyl all-trans-retinoate comprises from about 0.05% toabout 0.2% by weight of the composition.
 5. The method of claim 2wherein the vehicle is a mixture selected from the group consisting ofpropylene glycol-ethanol and propylene glycol-ethanol-chloroform.